Wolbachia genetically interacts with the bag of marbles germline stem cell gene in male D. melanogaster

The bacterial endosymbiont Wolbachia manipulates reproduction of its arthropod hosts to promote its own maternal vertical transmission. In female D. melanogaster , Wolbachia has been shown to genetically interact with three key reproductive genes ( bag of marbles ( bam ) , Sex-lethal, and mei-P26) , as it rescues the reduced female fertility or fecundity phenotype seen in partial loss-of-function mutants of these genes . Here, we show that Wolbachia also partially rescues male fertility in D. melanogaster carrying a new, largely sterile bam allele when in a bam null genetic background. This finding shows that the molecular mechanism of Wolbachia ’s influence on its hosts' reproduction involves interaction with genes in males as well as females, at least in D. melanogaster .


Description
The widespread bacterial endosymbiont Wolbachia is well known for its manipulation of host reproduction across arthropods. This includes rescue of fertility defects observed in partial loss-of-function mutants in D. melanogaster females, with genes including bag of marbles (bam) (Flores et al. 2015, Bubnell et al. 2021, Sex-lethal (Sxl) (Starr andCline 2002, Ote et al. 2016), and meiotic P26 (mei-P26) (Russell et al. 2022).
Bam is a germline stem cell (GSC) gene critical in female and male D. melanogaster where it regulates cell maintenance and differentiation. In females, bam functions primarily as a key switch gene that initiates the differentiation programming of GSC daughter cells into cystoblasts (McKearin and Spradling 1990). In males, bam functions to limit the mitotic divisions of spermatogonia and initiate programming of spermatocytes (McKearin and Spradling 1990, Gonczy 1997, Insco et al. 2009, Insco et al. 2012. In spermatogonia, Bam protein accumulates to a threshold that transitions the cells into spermatocytes. There, Bam is quickly downregulated and the translation of bam mRNAs is repressed by microRNAs (Eun et al. 2013).
It was previously shown that Wolbachia genetically interacts with bam in female D. melanogaster but not male D. melanogaster (Flores et al. 2015). However, because the bam mutant tested was sterile in males, it is unknown if the mutation is too severe for Wolbachia to rescue the fertility defect or if Wolbachia does not genetically interact with bam in males. For the former case, this possibility is evident in the lack of rescue of a bam null mutant (Flores et al. 2015). For the latter case, Wolbachia may not genetically interact with bam in males because of bam's differing function between the sexes.
We have found a bam allele that has reduced fertility in males when expressed as a transgene in a bam null background, thus allowing us to distinguish between the two possibilities. The bam allele is a PhiC31 generated transgene inserted at the attP40 site on the second chromosome and expresses a fluorescent Venus-tagged Bam protein. These flies have a severe fertility defect in a sensitized bam null background when uninfected with Wolbachia that is not seen in the transgenic bam line that lacks the Venus fluorescent tag in a similar bam null background (p<0.05) (Fig 1a).
We find that the reduced fertility of the transgenic bam::Venus males is rescued by Wolbachia (Fig 1a) (p<0.05). In contrast, the transgenic bam line without the Venus tag is not affected by Wolbachia infection. These results show that Wolbachia genetically interacts with bam in male D. melanogaster, as it does in female D. melanogaster.
We compared the cytology of this transgenic bam::Venus line to that of a single copy of an endogenous bam::Venus line that was generated with CRISPR (Bubnell 2020). The transgenic bam::Venus males displayed tumorous cells that continued to express Bam protein (Fig 1c). The GSC population in the testis hub appeared to remain intact with no obvious ectopic Bam expression in the stem cell niche. In contrast, the endogenous bam::Venus testes did not show a severe tumorous phenotype, though other cytological defects are observed, as bam is haploinsufficient in males and D. melanogaster testes require two copies of bam to be cytologically wildtype (Fig 1d) (Insco et al. 2009). These results suggest that the tumorous phenotype in the transgenic bam::Venus testes is due to the transgenic nature of the allele, wherein the genomic location of the transgene insert is likely affecting bam expression Rubin 1983, Levis et al. 1985). This is consistent with findings that reproduction is sensitive to bam expression levels in male D. melanogaster, as shown through cytological assays in Insco et al. (2009) and in a bam intron disruption line (Bubnell 2020).
Two lines of reasoning could explain Wolbachia's fertility rescue of our male hypomorphic mutant. The first is that it is evolutionarily advantageous for Wolbachia to inhabit a reproductively fit male host. Wolbachia spreads rapidly through populations by inducing cytoplasmic incompatibility, where Wolbachia manipulates host sperm (reviewed in Wang et al. 2022). Thus, it is not unreasonable to consider that, despite being maternally inherited, Wolbachia that help maintain proper spermatogenesis have been evolutionarily favored.
The second line of reasoning considers similarities in the germline of female and male D. melanogaster hosts. Wolbachia may interact with host proteins or RNA that function similarly in oogenesis and spermatogenesis. Thus, Wolbachia's genetic interaction with bam in males may be a byproduct of Wolbachia's actions in females that help maintain proper oogenesis to promote Wolbachia's survival through maternal transmission. In this vein, it is worth noting the recent report that Wolbachia also rescues the fertility defect of a largely sterile male mei-P26 null mutant (Russell et al. 2022), where mei-P26 is a gene critical for both male and female gametogenesis (Page et al. 2000;Neumuller et al. 2008).
Although bam's primary function is different in male and female D. melanogaster, there are some commonalities between males and females that we highlight here. 1) bam null mutants produce tumorous cells that display GSC-like qualities (in males, the tumorous cells also display some qualities of germ cells, such as incomplete cytokinesis and synchronic mitosis) (McKearin and Ohlstein 1995, Gonczy et al. 1997. 2) Ectopic Bam expression in GSCs can cause GSC loss (Ohlstein and McKearin 1997, Shulz et al. 2004, Kawase et al 2004, Flatt et al. 2008, Sheng et al. 2009. 3) Partially differentiated germ cells are able to dedifferentiate back into GSC-like cells with manipulation of bam (Kai andSpradling 2004, Sheng et al. 2009). And 4) bam may share a role of limiting mitotic divisions: it has been proposed that degradation of Bam after the fourth mitotic division prevents further mitosis in females (McKearin and Ohlstein 1995) and it has been shown in males that accumulation of Bam to a threshold stops the mitotic programming of spermatocytes (Insco et al. 2009).
Our current results do not allow us to distinguish between these two lines of reasoning to explain Wolbachia's fertility rescue of a male hypomorphic mutant and more research on bam function and its molecular partners in male D. melanogaster is needed before we are able to get a clear understanding of the similarities and differences in how Wolbachia interacts with bam in females and males. Nevertheless, establishing a genetic interaction between bam and Wolbachia in male D. melanogaster is an important insight into the functional interaction between Wolbachia, bam, and gametogenesis.

Methods
Fly lines. Two lines of transgenic flies were generated and examined for fertility defects in male D. melanogaster. These include the w; [w +mc bam::Venus] and a w; [w +mc bam] line that lacks the Venus fluorescent tag. The transgenic alleles were cloned and mutant flies were generated by PhiC31 transgenesis as described in detail in Wenzel and Aquadro (2023). Briefly, the entire bam coding region, its introns, and approximately 1.5kb upstream and 700bp downstream were cloned from the genomic DNA of a bam::Venus line (Bubnell 2020). The design of this construct is based on the D. melanogaster bam YFP line from Flores et al. (2015), but contains a 36bp glycine-glycine-serine linker sequence between the 3' end of the bam coding sequence and the Venus tag to promote proper protein folding. The bam::Venus construct was cloned into the pCasper\attB vector, a gift from Dan Barbash, and inserted at the attP40 site on the second chromosome, with plasmid injections performed by GenetiVision into the embryos of a w, nos-int; P{CaryP}attP40 D. melanogaster line.
Transgenic flies that were assessed for this study were in a null bam background such that the genotypes are w; [w +mc bam::Venus]/+; bam Δ59 /bam Δ86 and w; [w +mc bam]/+; bam Δ59 /bam Δ86 . bam Δ59 and bam Δ86 were originally obtained from Dennis McKearin. The wMel strain of Wolbachia was maternally crossed in from PCR-verified infected parental strains when appropriate during generation of the desired genotypes.
Fertility assays. Virgin transgenic males and virgin CantonS females were collected and aged four days at which time they were mated to each other in individual crosses. At least 29 crosses of one male and two females were set up on yeast-glucose food (minimum of 29, maximum of 40 crosses were set per genotype tested). All crosses were kept in an incubator at 25°C with a 12-h light/dark cycle. Parents were removed on the seventh day of mating. Progeny were counted every other day over seven days, starting on the day of first eclosion.
To offer greater transparency of data, estimation statistics and plots were generated using the dabestr package in R (v0.3.0) (Ho et al. 2019). The associated estimation statistics website was used to report p-values from two-sided permutation t-tests, as they were not provided in the R package (Claridge-Chang and Ho, accessed 2022 Sept 20).
Immunostaining. Immunostaining was performed on testes of 2-3 day old transgenic flies as described in Flores et al. (2015) and Bubnell et al. (2022). Briefly, testes were dissected in ice-cold 1x PBS and fixed in 4% paraformaldehyde (Electron Microscopy Sciences) for 15 min. Tissue was subsequently rinsed and washed with PBST (1X PBS, 0.1% Triton-X100), blocked in PBTA (1X PBS, 0.1% TritonX100, 3% BSA) (Alfa Aesar), and incubated overnight with primary antibodies. Rinse and wash steps were repeated with PBTA, followed by an overnight incubation with goat anti-serum, and a 2 hour incubation with secondary antibodies. Tissue was rinsed and washed again with PBST and mounted in ProLong Diamond AntiFade (Invitrogen).